Throughout this application, various publications are referenced by author and date within the text. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding the claims. All patents, patent applications and publications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
This invention relates to antiretroviral drug susceptibility and resistance tests to be used in identifying effective drug regimens for the treatment of human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS). The invention further relates to the means and methods of monitoring the clinical progression of HIV infection and its response to antiretroviral therapy using phenotypic or genotypic susceptibility assays. The invention also relates to novel vectors, host cells and compositions for carrying out phenotypic susceptibility tests. The invention further relates to the use of various genotypic methodologies to identify patients whose infection has become less susceptible (xe2x80x9cresistantxe2x80x9d) to a particular antiretroviral drug regimen. This invention also relates to the screening of candidate antiretroviral drugs for their capacity to inhibit viruses, selected viral sequences and/or viral proteins. More particularly, this invention relates to using phenotypic susceptibility tests and/or genotypic tests to identify patients whose virus/viruses exhibit drug-dependent stimulation of replication in the presence of anti-retroviral agents.
HIV infection is characterized by high rates of viral turnover throughout the disease process, eventually leading to CD4 depletion and disease progression (Wei X, Ghosh S K, Taylor M E, et al. (1995) Nature 343, 117-122) (Ho D D, Naumann A U, Perelson A S, et al. (1995) Nature 373, 123-126). The aim of antiretroviral therapy is to achieve substantial and prolonged suppression of viral replication. Achieving sustained viral control is likely to involve the use of sequential therapies, generally each therapy comprising combinations of three or more antiretroviral drugs. Choice of initial and subsequent therapy should, therefore, be made on a rational basis, with knowledge of resistance and cross-resistance patterns being vital to guiding those decisions. The primary rationale of combination therapy relates to synergistic or additive activity to achieve greater inhibition of viral replication. The tolerability of drug regimens will remain critical, however, as therapy will need to be maintained over many years.
In an untreated patient, some 1010 new viral particles are produced per day. Coupled with the failure of HIV reverse transcriptase (RT) to correct transcription errors by exonucleolytic proofreading, this high level of viral turnover results in 104 to 105 mutations per day at each position in the HIV genome. The result is the rapid establishment of extensive genotypic variation. While some template positions may be more error prone, (Mansky L M, Temin H M (1995) J Virol 69, 5087-5094) (Schinazi R F, Lloyd R M, Ramanathan C S, et al. (1994) Antimicrob Agents Chemother 38, 268-274), mathematical modeling suggests that, at every nucleotide position, mutation may occur 104 times per day in infected individuals.
For antiretroviral drug resistance to occur, the target enzyme must be modified while preserving its function in the presence of the inhibitor. Point mutations leading to an amino acid substitution may result in changes in shape, size, or charge of the active site, substrate binding site, or surrounding regions of the enzyme. Mutants resistant to antiretroviral agents have been detected at low levels before the initiation of therapy (Mohri H, Singh M K, Ching W T W, et al. (1993) Proc Natl Acad Sci USA 90, 25-29) (Nxc3xa1jera I, Richman D D, Olivares I, et al. (1994) AIDS Res Hum Retroviruses 10, 1479-1488) (Nxc3xa1jera I, Holguin A, Quixc3x1ones-Mateu E, et al. (1995) J Virol 69, 23-31). However, these mutant strains represent only a small proportion of the total viral load and may have a replication or competitive disadvantage compared with wildtype virus (Coffin J M (1995) Science 267, 483-489). The selective pressure of antiretroviral therapy provides these drug-resistant mutants with a competitive advantage and thus they come to represent the dominant quasispecies (Frost S D W, McLean A R (1994) AIDS 8, 323-332) (Kellam P, Boucher C A B, Tijnagal J M G H (1994) J Gen Virol 75, 341-351) ultimately leading to drug resistance and virologic failure in the patient.
A mutation or mutations that results in virus that can not only replicate in the presence of drug (i.e. resistant virus) but could actually replicate more efficiently in the presence of drug than in the absence of drug (i.e. drug-dependent stimulation of virus), would present an especially important phenotype to identify. In this case, a drug could actually accelerate the rate of destruction to the immune system and progression of disease.
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are a chemically diverse group of compounds which are potent inhibitors of HIV-1 Reverse Transcriptase (RT) in vitro. These compounds include pyridinone derivatives, bis(heteroaryl) piperazines (BHAPs) such as delavirdine and atevirdine, the dipyridodiazepinone (nevirapine), the thymine derivative groups (TSAO and HEPT), an a-anilino phenylacetamide (a-APA) compound (loviride), the quinoxaline-class inhibitors (HBY-097), the benzodiazepin-one and -thione (TIBO) compounds, and the pyridinone derivatives (L-697,661). For overviews, see (DeClercq E. (1996) Rev Med Virol 6, 97-117) (Emini E A (1996) Antiviral Drug Resistance, ed. D D Richman, John Wiley and Sons, Ltd). Three NNRTIs: nevirapine (NVP, Viramune, Boehringer Ingelheim, Ingelheim am Rhein, Germany), delavirdine (DLV, Rescriptor, Pharmacia and Upjohn, Kalamazoo, Mich., USA), and efavirenz (EFV, Sustiva, Dupont, Wilmington, Del., USA) are licensed for use in the USA.
High-level resistance to individual compounds appears to develop rapidly, often within a few weeks of initiating monotherapy, frequently involving only single-point mutations, and in many cases leading to considerable cross-resistance to other NNRTIs. Most mutations reported occur in the codon groups 100-108 and 181-190 which encode for the two b-sheets adjacent to the catalytic site of the RT enzyme (Kohlstaedt L A, Wang J, Friedman J M, et al. (1992) Science 256, 1783-90). The NNRTI binding pocket, as it has been described, is a hydrophobic non-substrate binding region of RT where these agents directly interact with RT. They inhibit activity by interfering with mobility of the xe2x80x98thumbxe2x80x99 subdomain, or disrupting the orientation of conserved aspartic acid side chains essential for catalytic activity (D""Aquilla R T. (1994) Clin Lab Med 14, 393-423) (Arnold E., Ding J., Hughes S H, et al. (1995) Curr Opin Struct Biol 5, 27-38).
Mutations conferring reduced susceptibility to nevirapine have been described at HIV RT codons 98, 100, 103, 106, 108, 181, 188 and 190 (Richman D D, Havlir D, Corbeil J. (1994) J Virol 68, 1660-1666). The most frequently selected variant during nevirapine monotherapy is a Tyr181àCys (Y181C) mutation, which results in a 100-fold reduction in sensitivity to this agent, and with reduced susceptibility to the pyridinone derivatives (L-696,229 and L-697,661) (Arnold, Ibid). TSAO also has limited activity in the presence of the Y181C mutation, but maintains activity in the presence of mutations HIV RT at codons 100 and 103, and in vitro selects for a unique mutation, GLU138àLys (E138K), in the region where it most closely interacts with RT (Richman, D D, Ibid) (Richman D D, Shih C K, Lowy I, et al. (1991) Proc Natl Acad Sci USA 88, 11241-11245).
Resistance to loviride when used as monotherapy develops in most patients by week 24. It has been mapped to a range of HIV RT codons 100-110; 181-190), most commonly codon 103 (Staszewski S, Miller V, Kober A, et al. (1996) Antiviral Ther 1, 42-50). During combination therapy using loviride with zidovudine or zidovudine plus lamivudine, variants at codons 98 and 103 were the most frequent mutations detected at 24 weeks (Staszewski S, Miller V, Rehmet S, et al. (1996) AIDS 10, F1-7).
Although the K101E, K103N, and Y181C, mutations also confer cross-resistance to BHAPs, (Balzarini J, Karlsson A, Pxc3xa9rez-Pxc3xa9rez M-J, et al. (1992) Virology 192, 246-253) the characteristic P236L substitution selected for by these agents in vitro appears to sensitize RT to some other NNRTIs, reducing the IC50 for nevirapine, for example, 7- to 10-fold, without influencing sensitivity to nucleoside analogues (Staszewski S., Ibid). This mutation at codon 236 has been observed in clinical isolates during atevirdine therapy, although other resistance-conferring mutations at codons 103 and 181 have been reported during monotherapy as well as at codons 101, 188, 233 and 238 during combination therapy with zidovudine.
While HBY-097 may initially select for a mutation at HIV RT codon 190 in vitro, further passage consistently selects for mutations at HIV RT codons 74 and 75, with some mutant viruses showing decreased sensitivity to didanosine and stavudine, but not zidovudine (Kleim J-P, Rxc3x6sner M, Winkler I, et al. (1995) J Acquir Immune Defic Syndr 10 Suppl 3, 2).
Mutation at codon 181 has been reported to antagonize zidovudine resistance due to the typical 41 and 215 codon mutations, (Zhang D, Caliendo A M, Eron J J, et al. (1994) Antimicrob Agents Chemother 38, 282-287) suggesting that combination therapy with some NNRTIs and zidovudine may be feasible. Although an HIV mutant with triple resistance to zidovudine, didanosine and nevirapine has been described in vitro, (Larder B A, Kellam P, Kemp S D (1993) Nature 365, 451-453) treatment with this triple combination does provide superior immunological and virological responses than treatment with zidovudine plus didanosine alone over a 48-week period in patients with CD4 cell counts  less than 350/mm3.
Combination therapy with zidovudine and the pyridinone derivative L-697,661 prevents the appearance of the codon 181 mutation typically selected during monotherapy with this NNRTI, delaying the appearance of high-level resistance to this compound. Changes in susceptibility to zidovudine were not examined in this study (Staszewski S, Massari F E, Kober A, et al. (1995) J Infect Dis 171, 1159-1165). Concomitant or alternating zidovudine therapy does not delay the appearance of resistance during nevirapine therapy (Richman D D, Ibid) (Nunberg J H, Schleif W A, Boots E J, et al. (1990) J Virol 65, 4887-4892) (DeJong M D, Loewenthl M, Boucher C A B, et al. (1994) J Infect Dis 169, 1346-1350) (Cheeseman S H, Havlir D, McLaughlin M M, et al. (1995) J Acquir Immune Defic Syndr 8, 141-151. However, the 181 mutant is not being observed during treatment with this combination, and the most common mutation occurs at 190 (Richman D D, Ibid). This suggests that the codon 181 mutation, which is antagonistic to zidovudine resistance in vitro, is not compatible, or not preferred in vivo, with selection favoring other mutations which allow for reduced susceptibility to nevirapine concomitant with zidovudine resistance.
The rapid development of reduced susceptibility to the NNRTIs suggests limited utility of these agents, particularly as monotherapies, and has led to the modification of these molecules in an attempt to delay the appearance of drug-resistant virus. A xe2x80x98second generationxe2x80x99 NNRTI, the pyridinone derivative L-702,019, demonstrated only a 3-fold change in IC50 between wild-type and codon 181 mutant HIV-1, and required multiple mutations to engender high-level resistance (Goldman M E, O""Brien J A, Ruffing T L, et al. (1993) Antimicrob Agents Chemother 37, 947-949).
Similarly, Efavirenz (EFZ) was introduced as a second generation NNRTI relatively recently. Efavirenz has a unique profile in that it retains activity against viruses containing the common RT mutation, Y181C. In vitro, efavirenz selects for mutations at codons 100, 101, 103, 108, 179, 181, and 188. This is similar to the in vivo resistance profile, which includes mutations at codons 100, 103, 108, 190 and 225, (and possibly 101, 179, 181 and 188). (Winslow D L, Garber S, Reid C, it al. Fourth International Antiviral Therapy 1977; 1(Suppl.1): 6. Conference on HIV Drug Resistance Sardinia, Italy, (1995) (Winslow D L, Garber S, Reid C, et al. Antiviral Therapy 1997; 1(suppl.1):6) (Young S D, Britcher S F, Tran L O, et al. Antimicrobial Agents and Chemotherapy 1995; 39.2602-2609.) (Bacheler L T, Anton E, Jeffrey S, et. al. Antiviral Therapy 1998; 3(Suppl.1): 15-16) (Bacheler L T, Weislow O, Snyder S and Hanna G. 12th World AIDS Conference, 1998, Geneva, Switzerland, Abstract 41213.)
It is an object of this invention to provide a drug susceptibility and resistance test capable of showing whether a viral population in a patient is resistant to a given prescribed drug. Another object of this invention is to provide a test that will enable the physician to substitute one or more drugs in a therapeutic regimen for a patient that has become resistant to a given drug or drugs after a course of therapy. Yet another object of this invention is to provide a test that will enable selection of an effective drug regimen for the treatment of HIV infections and/or AIDS. Yet another object of this invention is to provide the means for identifying the drugs to which a patient has become resistant, in particular identifying resistance to non-nucleoside reverse transcriptase inhibitors. (NNRTIs) Still another object of this invention is to provide a test and methods for evaluating the biological effectiveness of candidate drug compounds which act on specific viruses, viral genes and/or viral proteins particularly with respect to viral drug resistance associated with non-nucleoside reverse transcriptase inhibitors (NNRTIs). It is also an object of this invention to provide the means and compositions for evaluating HIV antiretroviral drug resistance and susceptibility. Still another object of this invention is to provide a means of determining whether a candidate anti-retroviral drug will cause increased or stimulated viral replication. This and other objects of this invention will be apparent from the specification as a whole.
The present invention relates to methods, using phenotypic and genotypic methods to monitor the clinical progression of human immunodeficiency virus infection and its response to antiviral therapy. The invention is also based, in part, on the discovery that genetic changes in HIV reverse transcriptase (RT), which confer resistance to antiretroviral therapy, may be rapidly determined directly from patient plasma HIV RNA using phenotypic or genotypic methods. The methods utilize polymerase chain reaction (PCR) based assays. Alternatively, methods evaluating viral nucleic acid or viral protein in the absence of an amplification step could utilize the teaching of this invention to monitor and/or modify antiretroviral therapy. This invention is based in part on the discovery of a mutation at codon 230 either alone or in combination with a mutation at codon 103 or 181 of HIV RT in NNRTI inhibitor treated patients, in which the presence of the mutations correlates with decreased susceptibility to delavirdine, nevirapine and efavirenz, and with drug-dependent stimulation of viral replication in the presence of delavirdine, nevirapine or efavirenz. The mutations were found in plasma HIV RNA after a period of time following initiation of therapy. The development of the mutation at codon 230, in addition to the mutation at codon 103 or 181 in HIV RT, was found to be an indicator of the development of resistance, and ultimately of immunological decline. Resistance test vectors containing the single site mutation at codon 230 (M230L), and M230L in combination with a mutation at either 103 (K103N) or 181 (Y181C) in reverse transcriptase were constructed using site directed mutagenesis (Sarkar G, Sommer S S. (1990). Biotechniques 8:404-407). These mutations were observed to be associated with decreased susceptibility to the NNRTI and, in some combinations, drug-dependent stimulation of viral replication.
This invention is based in part on the discovery of a mutation at codon 230 in combination with mutations at codons 101, 103, 190, 221 and 238 of HIV RT in NNRTI treated patients, in which the presence of the mutations correlates with a decrease in susceptibility to delavirdine, nevirapine and efavirenz, and with drugdependent stimulation of viral replication in the presence of delavirdine, nevirapine or efavirenz.
This invention is based in part on the discovery of a mutation at codon 241 of RT that was discovered to occur in NNRTI-treated patients. The presence of the mutation at 241, in addition to other NNRTI-resistance mutations (these mutations may include previously described NNRTI-resistance mutations such as: K101E, K103N, V106M, I135T, E138A and G190A) correlates with decreased susceptibility to delavirdine, nevirapine and efavirenz. Resistance test vectors containing patient sequences with these mutations exhibited reduced susceptibility to delavirdine, nevirapine and efavirenz as well as drug dependent stimulation of replication in the presence of all three drugs.
This invention is based in part on the discovery of mutations at codon 245 of RT that was discovered to occur in NNRTI-treated patients. The presence of the mutation at 245, in addition to other NNRTI-resistance mutations (which may include previously described NNRTI-resistance mutations such as: A98G, K101E, K103N, I135T, E138A, Y181C, G190A and P225H) correlates with decreased susceptibility to delavirdine, nevirapine and efavirenz. Resistance test vectors containing patient sequences with these mutations exhibited reduced susceptibility to delavirdine, nevirapine and efavirenz as well as drug dependent stimulation of replication in the presence of all three drugs. Resistance test vectors containing a single site mutation at codon 245 (V245E or T), and as well as test vectors containing V245E or T in combination with mutations at 103 (K103N) and 135 (I135T) in RT were constructed using site directed mutagenesis. While V245E alone had no effect on susceptibility to the NNRTI, The triple combination of mutations (K103N, I135T and 245 E or T) was observed to be associated with decreased susceptibility to the NNRTI and drug-dependent stimulation of viral replication.
This invention is based in part on the discovery of a mutation at codon 270 of RT that was discovered to occur in NNRTI-treated patients. The presence of the mutation at 270 in addition to other NNRTI-resistance mutations (which may include previously described NNRTI-resistance mutations such as: K103N, I135T and P225H) correlates with decreased susceptibility to delavirdine, nevirapine and efavirenz, and drug-dependent stimulation of viral replication. This invention is based in part on the discovery of a patient-derived segment containing multiple mutations at HIV RT codons 35, 67, 69, 70, 106, 189, 200, 202, 208, 211, 215, 218, 219, 221, 227, 228, 283, 284, 286, 293 and 297 of RT that was discovered in an NNRTI-treated patient. Resistance test vectors containing patient sequences with these mutations exhibited reduced susceptibility to delavirdine, nevirapine and efavirenz as well as drug dependent stimulation of replication in the presence of all three drugs. Site-directed reversion of specific mutations demonstrated that many of the mutations play a role in the drug-dependent stimulation of viral replication, but that none of the mutations is sufficient on it""s own to cause such an effect. Specifically, mutations at 106, 189, 227, 283, 284 and 286 are observed to modulate the resistance and stimulation of viral replication seen with this.